Through all kingdoms of life RNAs are modified as, or after, they are synthesized. The types and sites of modification are often conserved, implying conservation of function. Approximately 0.8% of the coding capacity of the E. coli cell is dedicated to encoding enzymes that modify RNA. Many of these modifications are located at key functional regions of the ribosome or other RNAs. The enzymes that carry out the modifications exhibit unique or limited multisite specificity. The aim of this proposal is to determine the basis for selectivity and catalysis in three families of RNA modifying enzymes: 5-methyl uracil (m5U) methyltransferases (MTases), 5-methyl cystosine (mSC) MTases, and pseudouridine synthases (Psi synthases). These families evoke some of the most common conserved modifications of the more than 80 different types seen in RNA. A common feature of the chemical mechanisms of these families is Michael addition of an amino acid nucleophile to C-6 of the target pyrimidine to activate C-5 for alkylation. Each modification is evoked at a particular stage in RNA folding suggesting that the determinants of selectivity often involve unique three-dimensional folded structures. We seek to determine X-ray crystal structures of several members of each family to elucidate the basis for three-dimensional selectivity in enzyme-RNA targeting. The strategy is to express each enzyme from E. coli and orthologs from two other species, B. subtilis and T. maritima, to provide many candidates for crystallization trials. A minimal, often 17-50 base RNA substrate is sought. A synthetic minimal substrate, containing 5-fluorofoyrimidine at the target site, when reacted with the protein yields a covalent protein-substrate complex, providing an effective means to crystallize enzyme-RNA complexes. The structures of these are compared to structures of enzymes alone or with cofactors. The structures are the basis for the design of mutations in the enzyme or the substrate, and for computational evaluations. Biochemical and structural analysis of the mutant enzymes or substrates further elucidate contributors to the high selectivity and enzyme mechanism. An unrelated RNA MTase, human m1A58 tRNA MTase, will also be subject to structural determination to facilitate structure-based drug discovery. This is a required enzyme in HIV replication, and as such it may represent a new target for anti-HIV drugs.